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Open AccessArticle

Simultaneous Ligand and Receptor Tracking through NMR Spectroscopy Enabled by Distinct 19F Labels

1
Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada
2
Institut de Pharmacologie, Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada
3
Department of Pharmacology-Physiology, Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada
4
Department of Chemistry, Dalhousie University, Halifax, NS B3H 4R2, Canada
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(15), 3658; https://doi.org/10.3390/ijms20153658
Received: 3 July 2019 / Revised: 19 July 2019 / Accepted: 23 July 2019 / Published: 26 July 2019
(This article belongs to the Special Issue Structure and Function of Membrane Proteins)
To probe ligand-receptor binding at the atomic-level, a frequent approach involves multidimensional nuclear magnetic resonance (NMR) spectroscopy experiments relying on 13C- and/or 15N-enrichment alongside 1H. Alternatively, the lack of fluorine in biomolecules may be exploited through specific incorporation of 19F nuclei into a sample. The 19F nucleus is highly sensitive to environmental changes and allows for one-dimensional NMR spectroscopic study, with perturbation to chemical shift and spin dynamics diagnostic of structural change, ligand binding, and modified conformational sampling. This was applied to the apelinergic system, which comprises a rhodopsin-like G protein-coupled receptor (the apelin receptor (AR)/APJ) and two families of cognate ligands, the apelin and apela (ELABELA/toddler) peptides. Specifically, AR fragments consisting of either the N-terminal tail and first transmembrane (TM) α-helix (AR55) or the first three transmembrane α-helices (TM1-3) were prepared with biosynthetic fluorotryptophan incorporation. Interactions of each AR fragment with a high-affinity, 2,4,5-trifluorophenylalanine labeled apelin analogue were compared by 19F NMR. Distinct ranges of 19F chemical shifts for ligand and receptor provide unambiguous tracking of both species, with distinct binding behaviour observed for each AR fragment implying that AR55 is not sufficient to recapitulate the physiological binding event. Site-specific perturbation was also apparent for the apelin analogue as a function of substitution site, indicating an orientational binding preference. As a whole, this strategy of distinctive 19F labelling for ligand and receptor provides a relatively fast (i.e., employing 1D NMR experiments) and highly sensitive method to simultaneously and definitively track binding in both species. View Full-Text
Keywords: 19F NMR; G protein-coupled receptor (GPCR); apelin receptor (APJ); ligand-receptor interactions; peptide-GPCR binding; 19F-labelled apelin analogues 19F NMR; G protein-coupled receptor (GPCR); apelin receptor (APJ); ligand-receptor interactions; peptide-GPCR binding; 19F-labelled apelin analogues
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Simmons, J.R.; Murza, A.; Lumsden, M.D.; Kenward, C.; Marsault, É.; Rainey, J.K. Simultaneous Ligand and Receptor Tracking through NMR Spectroscopy Enabled by Distinct 19F Labels. Int. J. Mol. Sci. 2019, 20, 3658.

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